In processes requiring the separation and analysis of biomolecules such as proteins, nucleic acids, lipids, and carbohydrates, it is often convenient to bind the biomolecules to a solid matrix at some point in the process flow. Such binding allows materials that might be troublesome in further analysis to be removed while retaining the biomolecule or biomolecules of interest. As an example, a well-known technique for separating proteins in a protein mixture is by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, known in the art as SDS-PAGE. A potential problem in the overall procedure is that SDS and other materials in the sample after electrophoresis can interfere with subsequent analysis. If the proteins can be bound to a support, the SDS and other unwanted chemicals or reagents can be relatively easily removed. A sample support with the sample proteins bound thereto is also convenient for further handling of sample in analytical techniques such as, for example, amino acid and sequence analysis. To be useful, such a support needs to be compatible with the organic solvents, acids, and bases used in amino acid analysis and sequencing.
A variety of support matrices and attachment chemistries have been developed in the art, depending on the particular biomolecule to be separated. These materials include, for example, nitrocellulose, DEAE-cellulose, derivatized glass beads, derivatized nylon, parchment, macrocyclic polyethers, polyvinylbutyral resin, polyvinylalcoholcollagen, polyvinylidene difluoride, and other polymers. As a specific example, diisothiocyanate (DITC) derivitized glass beads have been used as well as DITC functionalized glass fiber sheets. These matrices both react with the epsilon-amino groups of the lysine side chains of peptides and proteins. Subsequent analysis of proteins resolved by gel electrophoresis can be performed by blotting or electroblotting directly onto DITC-activated glass fiber sheets with covalent attachment occurring during the transfer. Disadvantages are that the glass fiber sheets are fragile in subsequent handling, and it is difficult to detect the protein molecules on the glass surface. More importantly, however, is that they have a relatively low protein binding capacity.
More recently polyvinylidene difluoride (PVDF) membranes have been used in blotting and electroblotting, and have proved to be relatively more useful than glass supports. The membrane bound molecules can be readily visualized with a variety of compound, for example, by staining, such as with coomassie blue for proteins. Also PVDF membranes with bound proteins can be used as supports in state-of-the-art protein sequencing equipment, because the PVDF material is compatible with the materials and conditions encountered in such equipment. Furthermore, the binding to PVDF is typically reversible, so that the bound molecules can be easily removed from the membrane for subsequent analysis.
Even more recently the DITC attachment chemistry has been combined with PVDF membranes to produce membranes with even better properties for binding of biomolecules than PVDF alone. An example is the Immobilon.TM. PVDF Transfer Membrane developed and sold by Millipore Corporation of Bedford MA, which is a hydrophobic membrane having generally a broad affinity for proteins. Another membrane, Immobilon N.TM. PVDF Transfer Membrane, also developed and sold by Millipore Corporation, is derivatized to be hydrophilic and hence is used primarily as a DNA binding substrate. Other examples of some of the more recent developments include such membranes as ProBlott.TM. available from Applied Biosystems (another PVDF membrane), and derivatized nitrocellulose. Although these membranes have emerged as preferred membranes for electroblotting/sequencing of different kinds of biomolecules, there are still several problems that can cause low recoveries and variable results. The problems most often encountered during electroblotting or in other sample adsorption techniques onto these membranes include a variability in electrotransfer for different proteins, long times required (as long as 24 hours ) for adsorption of molecules onto the membranes from solution, variable results in recovery due to selectivity, and staining/destaining methods which reduce sequencing yields. The blotting techniques, moreover, are not very useful for small amounts of sample and sample in free solution.
What is clearly needed is a method and apparatus for binding biomolecules to solid supports that is useful for small amounts of sample in large volumes; that is rapid; that is not dependent on electrotransfer and does not exhibit the variability observed in electroblotting; that, for proteins is not selective (as shown in electroblotting); and is not dependent on staining techniques which cause sample loss (ie. to find the sample).